MO_II_L: Molecular Sciences II. Lectures
Áron Pánczél1, Attila Mócsai1
1 Semmelweis University, Faculty of Medicine, Department of Physiology
Introduction • Osteoclasts are multinucleated syncytia of myeloid origin responsible for bone resorption under various physiological and pathological conditions such as osteoporosis, bone-metastasis or autoimmune arthritis. Though they can be readily differentiated in vitro, the quantitative evaluation of such experiments is at present rather challenging.
Aims • The objective of our study was to develop fluorescence-based methods for efficient, real-time monitoring of the development of osteoclast cultures.
Methods • Our osteoclastogenesis assays are based on two genetic modifications in mice. The cathepsin K cre (Ctskcre) mutation results in the osteoclast-specific expression of the cre recombinase, while the mTmG mutation is a cre-responsive reporter switching from red to green fluorescence upon recombination.
Results • First we crossed Ctskcre and mTmG-carrying mice to obtain a Ctsk-reporter strain. This resulted in robust green signals in osteoclast cultures. Green fluorescence emerged in both mononuclear pre-osteoclasts and multinuclear osteoclasts. Then, we set up a second assay where Ctskcre cells were co-cultured with mTmG cells as a fusion-specific reporter. Here only upon cell-to-cell fusion could cre access its mTmG substrate and thus activate green fluorescence. This time a less robust green fluorescence could be detected that seemed to be confined to multinuclear syncytia but not to mononuclear cells. Parallel macrophage cultures, as well as osteoclast cultures lacking either the Ctskcre or the mTmG mutation, were devoid of green fluorescence indicating the osteoclast-specific nature and confirming the genetic working principle of both assays. We also demonstrated the underlying mechanics of our assays at genomic DNA, transcriptional and protein levels.
Conclusion • In summary, we successfully established two fluorescence based assays that enabled us to follow real-time changes in cell cultures differentiated towards osteoclasts. The included control cultures confirm a high degree of specificity for osteoclasts while the possibility of automated quantification highlights the potential of our approach for high-throughput (e.g. drug-screening) scenarios.
Funding • This work was supported by the Hungarian National Scientific Research Fund (NKFIH-OTKA No. K119653 and NVKP_16-1-2016-0039).
Semmelweis University, Doctoral School of Molecular Medicine